Most human genes have a "split" structure with exon segments (sense sequences) interrupted by intron sequences (nonsense sequences). The nuclear splicing process responsible for precise excision of the intron sequences and the joining of the exon sequences is critical for the viability of all known multicellular organisms. More importantly, regulation of this splicing process, so that different combinations of exons are assembled from the same set of gene sequences in different cell types, is central to development and cancer. Significant progress has been made in the study of the spliceosome process active in the splicing of pre-mRNA. However, the two fundamental questions in the field remain unanswered at least for mammalian systems. These are the long term objectives of the proposal: (a) the nature of the catalytic processes responsible for breaking and joining RNA and (b) the nature of recognition of RNA sequences in the pre-mRNA responsible for selection of splice sites. These broad and long term objectives are focused on four short term aims all of which grow from current research activities in the laboratory. (1) Novel methods based on site-specific crosslinking of RNA to either proteins or RNA will be used to probe the spliceosome and other complexes for components in contact with RNA. (2) The role in promoting splicing in reactions depleted of U1 snRNP of a highly evolutionarily conserved family of proteins which contains tracts of Ser-Arg (SIR) repeats will be studied. This reaction will be used to study recognition of the consensus sequence at 5' splice site within the spliceosome and potentially the sequence specific recognition of pre-mRNA by the SIR proteins. (3) Monoclonal antibodies specific for nuclear matrix which stain interphase cells in a "speckled" fashion will be used to study the potential relationship between nuclear matrix, S/R proteins and RNA splicing. (4) Cellular factors which recognize a RNA repeated sequence in an intron adjacent to the EIIIB exon of the fibronectin gene and regulate the splicing of this exon in a cell-type specific fashion will be identified and studied.